Beruflich Dokumente
Kultur Dokumente
77
appressoria that are typical for many plant pathogenic fungi (reviewed by Mendgen et al.,
1996). Several authors observed the swelling of hyphal tips of germ tubes and interpreted
these as an appressorium-like structure (Akutsu et al., 1981; van den Heuvel and
Waterreus, 1983; Cole et al., 1996). Recent microscopic and histochemical studies
(Tenberge et al., 2002) and gene function analysis (Gourgues et al., 2003) indicate that
these structures act as functional appressoria. The swelling of the hyphal tip may be the
consequence of a rise in osmotic value in the hyphal tip, resulting in water absorption. In
the absence of a rigid layer in the outer wall, swelling cannot result in an equally high
turgor as in the appressoria of Magnaporthe grisea (Howard et al., 1991; de Jong et al.,
1997). The extracellular matrix may contribute to the swelling by retaining water, as its
major polysaccharide component (cinerean), is extremely hygroscopic.
Penetration of the Host Surface
Invasion of host tissue can be achieved by active penetration or passive ingress. B.
cinerea is an opportunist that can initiate infection at wound sites, or at sites previously
infected by other pathogens. B. cinerea can also enter the substomatal cavity via an open
stoma. Nevertheless the pathogen is perfectly able to penetrate intact host surfaces. Only
direct penetration of the epidermal surface is discussed in this paragraph. For reasons of
simplicity the penetration of dead or wounded tissue, as well as via stomata, is regarded
as an expansion rather then a penetration process, and is dealt with in later paragraphs.
The cuticle consists of cutin, a polyester of hydroxylated and epoxidized C16- and
C18-fatty acids, covered with wax. Physical damage or brute mechanical penetration of
the cuticle by B. cinerea is not usually observed (Williamson et al., 1995; Cole et al.,
1996). Hence, it was considered that enzymatic (cutinolytic) activity is required for
penetrating intact host surfaces (Salinas and Verhoeff, 1995; van der Vlugt-Bergmans,
1997a). Salinas (1992) raised monoclonal antibodies against a 18 kDa cutinase.
Application of the antibody to gerbera flowers prior to inoculation reduced the number of
lesions formed. The gene encoding this cutinase enzyme was cloned (van der VlugtBergmans et al., 1997a) and a gene replacement mutant was made that was entirely
devoid of this cutinase activity (van Kan et al., 1997). The mutant was equally virulent as
the wild type isolate, both on gerbera flowers and tomato fruits and the fungus remained
able to penetrate intact cuticle surfaces (van Kan et al., 1997). Although the observations
of Salinas (1992) remain to be explained, it can be concluded that this 18 kDa cutinase is
not essential in penetration.
A different enzyme that may mediate host penetration is a 60 kDa lipase
(Commnil et al., 1995), inducible by apple cutin (Commnil et al., 1998) as well as
grape berry cuticle components (Commnil et al., 1999). The lipase possesses cutinolytic
activity although with clearly distinct kinetic properties than the 'typical' cutinase
mentioned above (Commnil et al., 1998). When polyclonal antibodies raised against this
lipase were applied prior to inoculation with B. cinerea conidia, germ tubes were
prevented from penetrating the cuticle. The antibodies did not affect germination
(Commnil et al., 1998). The protein has been partially sequenced (Commenil et al.,
1999). Constructing a targeted lipase-deficient mutant and determining its virulence
should assess the role of the lipase in host tissue penetration.
Killing the Host
B. cinerea kills host cells before they are invaded by hyphae (Clark and Lorbeer,
1976). Invasion of plant tissue by B. cinerea triggers nuclear condensation and plant
membrane damage, indicators for programmed cell death, in a ring of cells around the
hyphae (Govrin and Levine, 2000). These results imply that diffusible factors have a
direct or indirect phytotoxic activity. These factors may be proteins or low molecular
weight compounds secreted by the fungus into its environment. The induction of
programmed cell death facilitates B. cinerea invasion and may in fact be essential for
successful infection (Govrin and Levine, 2000).
1. Toxins. Culture filtrates of B. cinerea may induce toxic effects when applied to plant
79
still unclear. Fungal extracellular sugar oxidases (Liu et al., 1998; Edlich et al., 1989) or
superoxide dismutase (Tenberge et al., 2002) may be responsible for generating the H2O2.
The identification of the fungal enzymes that are important for H2O2 accumulation at the
host-fungus interface awaits molecular-genetic studies with targeted mutants.
Formation of Primary Lesions, Defense Responses in the Host
Host surface penetration and the rupture of plant cell walls by enzymes of B.
cinerea triggers a cascade of processes in the fungus as well as the host. This paragraph
deals with defense responses at the host-fungus interface and their impact on the progress
of infection.
1. Induction of Necrosis. The initial establishment of primary necrotic lesions coincides
with (and is in fact the result of) host defense activation in the neighbouring tissue in
response to the death of an invaded cell. It is as yet unclear whether cell death caused by a
necrotroph, such as B. cinerea, is equivalent to cell death during a hypersensitive
response (HR) to a biotrophic pathogen (reviewed by Lamb and Dixon, 1997). Govrin
and Levine (2000) showed that an oxidative burst occurs in plant tissue several cell layers
away from the fungal hyphae. Cytological staining provided evidence for rapid nuclear
condensation and irreversible membrane damage, indicative of a programmed cell death
process (Govrin and Levine, 2000). Largely the same defense responses are activated
during an infection by B. cinerea as during HR to avirulent races of a biotrophic
pathogen: lignification (Maule and Ride, 1976; Heale and Sharman, 1977), biosynthesis
of phytoalexins (e.g. Bennett et al., 1994) and accumulation of PR proteins (e.g. Benito et
al., 1998; Diaz et al., 2002). The total spectrum of defense responses results in a primary
necrotic lesion in which the fungus is effectively restricted. Depending on the type of host
tissue and yet unidentified physiological aspects of the host, the lesions enter a lag phase
in which they do not expand. A proportion of the primary lesions eventually develops into
aggressive, expanding lesions (van den Heuvel, 1981; De Meyer and Hfte, 1997; Benito
et al., 1998; Diaz et al., 2002). In the non-expanding lesions the fungus is not killed, since
viable fungal mycelium could be recovered from all lesions (Benito and van Kan,
unpublished results). Thus, an active defense contributes to (temporarily) restricting the
fungus within the primary lesions, giving rise to a period of quiescence.
2. Quiescence. In some tissues, B. cinerea causes long-lasting quiescent infections
(reviewed by Prusky, 1996), in which no symptoms are discernible at first. Prominent
examples are described in soft fruit such as strawberry, raspberry and grape. In these
hosts, B. cinerea predominantly infects the host flowers and resides quiescent in the
developing fruit tissue, often for several weeks. Fungal growth resumes at the onset of
fruit ripening. It has been postulated that high levels of fungitoxic or fungistatic
compounds (phytoalexins) in immature fruits contribute to grey mould quiescence. The
level of these compounds decreases during the ripening process concomitant with fungal
outgrowth. Attempts have been undertaken to increase the levels of antifungal compounds
or to prevent their degradation during ripening. The level of the stilbene phytoalexin
resveratrol in grapes is correlated with grey mould resistance (Langcake, 1981; Bavaresco
et al., 1997). The effect of over-expressing stilbene synthase genes from Vitis in
transgenic plants on resistance towards B. cinerea was evaluated. A significant, partial
resistance was obtained in tobacco (Hain et al., 1993) but not in tomato (Thomzik et al.,
1997).
Besides phytoalexins, immature fruits usually contain high levels of proteinaceous
inhibitors of fungal cell wall degrading enzymes, the PolyGalacturonase Inhibiting
Proteins (PGIPs) and their level decreases during ripening (De Lorenzo et al., 2001). In
view of the significant role that polygalacturonases play in the infection (see below),
efforts to produce transgenic plants overexpressing PGIPs have been undertaken to obtain
resistance towards B. cinerea. Indeed high constitutive expression of a heterologous PGIP
gene in tomato and an endogenous PGIP gene in Arabidopsis resulted in an increased
resistance to B. cinerea (Powell et al., 2000; Ferrari et al., 2003). One of the
considerations in this strategy is that PGIPs have a differential activity towards individual
81
fungal endoPGs (Desiderio et al., 1997; De Lorenzo et al., 2001). This makes it relevant
to chose PGIPs that are most potent against the B. cinerea endoPG isozymes that are
important in virulence (Ten Have et al., 2002).
Evasion of Chemical Defense
Pathogenic fungi have developed mechanisms to overcome deleterious effect of
preformed (phytoanticipins) or induced (phytoalexins) plant defense compounds. One
such mechanism involves an energy-dependent secretion by ABC-transporters that
confers some degree of tolerance to the fungitoxic effects of such compounds (de Waard,
1997). About a dozen ABC transporters have been cloned from B. cinerea (de Waard,
unpublished results) and their role in resistance towards plant defense compounds and
virulence on various hosts is being studied (Schoonbeek et al., 2001; 2002).
The major strategy of plant pathogenic fungi to deal with antifungal plant
compounds is an enzymatic detoxification (reviewed by Osbourn et al., 1998). Because of
its broad host range, B. cinerea encounters a wide spectrum of antimicrobial compounds,
depending on the host species that it attempts to infect. The ability of B. cinerea to
degrade or detoxify phytoalexins was already intensively studied two decades ago
(reviewed by Mansfield, 1980). The best studied example for phytoalexin detoxification
by B. cinerea is the detoxification of the Vitis phytoalexins pterostilbene and resveratrol.
The ability of fungal isolates to detoxify these phytoalexins was correlated with their
virulence (Sbaghi et al., 1996). Conversely, the resistance level of Vitis genotypes against
grey mould is correlated with their phytoalexin content (Langcake, 1981; Jeandet et al.,
1992). B. cinerea produces a substrate-specific laccase (stilbene oxidase) that is able to
oxidize both compounds to non-toxic derivatives (Pezet et al., 1991). The enzyme was
purified and characterized (Pezet, 1998) and the structure of stilbene degradation products
elucidated (Breuil et al., 1998). Schouten et al. (2002b) cloned a laccase gene responsible
for resveratrol conversion and generated targeted mutants that lost the ability to convert
resveratrol. The mutants did not show reduced virulence on Vitis or any other host tested
(Schouten et al., 2002b), indicating resveratrol conversion is not essential for virulence.
B. cinerea is also able to detoxify preformed antimicrobial compounds such as the
tomato saponin -tomatine (Verhoeff and Liem, 1975). Quidde et al. (1998) showed that
-tomatine is only partly deglycosylated by removal of the terminal xylose, yielding 1tomatine which is less toxic than -tomatine. A field survey showed that B. cinerea
isolates from various host plants and geographic origin all possessed tomatinase activity,
with one exception. Interestingly, the strain lacking tomatinase activity was highly
sensitive to -tomatine, completely non-pathogenic on tomato, yet highly aggressive on
Phaseolus vulgaris (Quidde et al., 1998). These data suggest that the ability to detoxify tomatine is correlated with virulence of B. cinerea on tomato. However, this strain might
have further defects that cause the specific loss of virulence on tomato; the cloning and
targeted mutagenesis of the tomatinase gene in a wild type strain will be necessary for
final evaluation of the role of tomatinase in the infection of tomato.
The oxidative burst at the host-pathogen interface imposes stress on the host as
well as the pathogen (Schouten et al., 2002a). B. cinerea is able to cope with external
oxidative stress in order to survive in the necrotic tissue. Successful detoxification of
H2O2 is presumably mediated by an extracellular catalase (Schouten et al., 2002a) with a
Glutathione S-Transferase functioning as intracellular back-up (Prins et al., 2000a;
Schouten et al., 2002a). Targeted mutagenesis of the extracellular catalase gene did not
negatively affect the survival of the mutant within the oxidative environment of a necrotic
lesion. Virulence of the mutant on several hosts was indistinguishable from that of the
wild type (Schouten et al., 2002a).
Disease Expansion and Tissue Maceration
B. cinerea must be able to macerate plant tissue and convert it into fungal
biomass. The initial step in expansion of primary lesions is presumably the killing of
neighbouring cells by mechanisms similar to the ones described above. In order to grow
82
from the primary lesion into neighbouring tissue, B. cinerea must actively degrade plant
cells. The major barrier that the pathogen encounters is the host cell wall. Cell wall
degradation facilitates the entry of the pathogen and it provides nutrients for growth
(reviewed by Ten Have et al., 2002).
Microscopic studies showed that after penetration of the cuticle, hyphae of B.
cinerea frequently invade the anticlinal wall between two epidermal cells. The
concomitant swelling of the epidermal cell wall (Mansfield and Richardson, 1981) is
indicative for the degradation of pectin in the matrix of the epidermal wall, resulting in
water absorption. Biochemical evidence suggested that pectinases are involved in primary
infection. B. cinerea possesses a set of cell wall degrading enzymes (CWDEs) including
pectin lyase (Movahedi and Heale, 1990), pectin methylesterase (Reignault et al., 1994;
Valette-Collet and Boccara, 2003), exo- and endo-polygalacturonase (Johnston and
Williamson, 1992) and cellulase (Barkai-Golan et al., 1988). B. cinerea genes have been
cloned that encode CWDEs: pectin and pectate lyase (Mulder, unpublished),
rhamnogalacturonan-hydrolase (Chen et al., 1997) six endoPGs (Ten Have et al., 1998,
Wubben et al., 1999), pectin methylesterase (Valette-Collet et al., 2003) and cellulases
(van Kan et al., unpublished). The endoPG genes, denoted Bcpg1-6, constitute a well
studied and, most probably, complete gene family. The expression patterns of the
individual endoPG genes in planta depend on the host species that is infected, on the stage
of the infection, as well as on the external conditions during which infection occurs (Ten
Have et al., 2001).
Targeted deletion mutants were made in Bcpg1 by gene replacement. The lesion
expansion rate of such mutants was reduced by about 25% as compared to the wild type
(Ten Have et al., 1998). The BcPG1 protein might be important in facilitating
intercellular growth at the periphery of the invading hyphae. Another explanation for the
reduction in virulence may be purely nutritional. The absence of BcPG1 reduces the
release of pectin degradation products that serve as nutrients, hence resulting in slower
growth of the fungus through the tissue. Mutagenesis of four additional endoPG genes is
in progress (van Kan et al., unpublished results). The distinction between a pathogenic
function and a nutritional function of B. cinerea endoPGs (Ten Have et al., 2002) is
difficult to make.
SUMMARY AND CONCLUDING REMARKS
Molecular-genetic tools are available to validate microscopic and biochemical
observations of the infection strategy of B. cinerea. Dozens of genes have been cloned
and their expression in planta or in vitro studied. The role(s) of individual genes in the
infection process can be analysed by targeted mutagenesis and studying the behaviour of
mutants on various hosts. This overview demonstrates that the pathogen is versatile and
uses a combination of factors during pathogenesis. We are beginning to understand the
roles of various factors in the different stages of the disease cycle and the ways in which
some of these factors interact. There seems to be a delicate balance between the attack
mechanisms of the fungus and the defense of the host. Such knowledge will contribute to
new, rational disease control strategies. The future challenge lies in the design of methods
that alter the balance in the interaction in favor of the host. Such a strategy will likely
consist of a sophisticated combination of biological control agents, appropriate (partially)
resistant plant genotypes and chemicals that either enhance the plant defense response or
interfere with crucial steps in the infection process.
ACKNOWLEDGEMENTS
Several research projects in my group are supported by the Technology
Foundation (STW), subsidized by the Ministry of Economic Affairs and The Netherlands
Organization for Scientific Research (NWO). I am grateful to Jos Raaijmakers for critical
reading of the manuscript.
83
Literature Cited
Akutsu, K., Kobayashi, Y., Matsuzawa, Y., Watanabe, T., Ko, K. and Misato, T. 1981.
Morphological studies on infection process of cucumber leaves by conidia of Botrytis
cinerea stimulated with various purine-related compounds. Ann. Phytopathol. Soc.
Japan 47: 234-243.
Barkai-Golan, R., Lavy Meir, G. and Kopeliovitch, E. 1988. Pectolytic and cellulolytic
activity of Botrytis cinerea Pers. related to infection of non-ripening tomato mutants.
J. Phytopathol. 123: 174-183.
Barkai-Golan, R., Lavy Meir, G. and Kopeliovitch, E. 1989. Stimulation of fruit ethylene
production by wounding and by Botrytis cinerea and Geotrichum candidum infection
in normal and non-ripening tomatoes. J. Phytopathol. 125: 148-156.
Bavaresco, L., Petegolli, D., Cantu, E., Fregoni, M., Chiusa, G. and Trevisan, M. 1997.
Elicitation and accumulation of stilbene phytoalexins in grapevine berries infected by
Botrytis cinerea. Vitis 36: 77-83.
Benito, E.P., Prins, T.W. and Van Kan, J.A.L.. 1996. Application of differential display
RT-PCR to the analysis of gene expression in a plant fungus interaction. Plant Mol.
Biol. 32: 947-957.
Benito, E.P., Ten Have, A., Van 't Klooster, J.W. and Van Kan, J.A.L. 1998. Fungal and
plant gene expression during synchronized infection of tomato leaves by Botrytis
cinerea. European J. Plant Pathol. 104: 207-220.
Bennett, M.H., Gallagher, M.D.S., Bestwick, C.S., Rossiter, J.T. and Mansfield, J.W.
1994. The phytoalexin response of lettuce to challenge by Botrytis cinerea, Bremia
lactucae and Pseudomonas syringae pv. phaseolicola. Physiol. Mol. Plant Pathol. 44:
321-333.
Breuil, A.C., Adrian, M., Pirio, N., Meunier, P., Bessis, R. and Jeandet, P. (1998)
Metabolism of stilbene phytoalexins by Botrytis cinerea: I. Characterization of a
resveratrol dehydrodimer. Tetrahedron Letters 39: 537-540.
Chen, H.J., Smith, D.L., Starrett, D.A., Zhou, D.B., Tucker, M.L., Solomos, T. and Gross,
K.C. 1997. Cloning and characterisation of a rhamnogalacturonan hydrolase gene
from Botrytis cinerea. Biochem. Mol. Biol. Int. 43: 823-838.
Clark, C.A. and Lorbeer, J.W. 1976. Comparative histopathology of Botrytis squamosa
and B. cinerea on onion leaves. Phytopathology 66: 1279-1289.
Cole, L., Dewey, F.M. and Hawes, C.R. 1996. Infection mechanisms of Botrytis species:
Pre-penetration and pre-infection processes of dry and wet conidia. Mycol. Res. 100:
277-286.
Colmenares, A.J., Aleu, J., Duran, R.P., Collado, I.G. and Hernandez, R.G. 2002. The
putative role of botrydial and related metabolites in the infection mechanism of
Botrytis cinerea. J. Chem. Ecol. 28: 997-1005.
Commnil, P., Belingheri, L., Sancholle, M. and Dehorter, B. 1995. Purification and
properties of an extracellular lipase from the fungus Botrytis cinerea. Lipids 30: 351356.
Commnil, P., Belingheri, L. and Dehorter, B. 1998. Antilipase antibodies prevent
infection of tomato leaves by Botrytis cinerea. Physiol. Mol. Plant Pathol. 52: 1-14.
Commnil, P., Belingheri, L., Bauw, G. and Dehorter, B. 1999. Molecular
characterization of a lipase induced in Botrytis cinerea by components of grape berry
cuticle. Physiol. Mol. Plant Pathol. 55: 37-43.
Cutler, H.G., Jacyno, J.M., Harwood, J.S., Dulik, D., Goodrich, P.D. and Roberts, R.G.
1993. Botcinolide: a biologically active natural product from Botrytis cinerea. Biosci.,
Biotechnol. Biochem. 57: 980-1982.
De Meyer, G. and Hfte, M. 1997. Salicylic acid produced by the rhizobacterium
Pseudomonas aeruginosa 7NSK2 induces resistance to leaf infection by Botrytis
cinerea on bean. Phytopathology 87: 588-593.
Deighton, N., Muckenschnabel, I., Colmenares, A.J., Collado, I.G. and Williamson, B.
2001. Botrydial is produced in plant tissues infected by Botrytis cinerea.
Phytochemistry 57: 689-692.
84
de Jong, J.C., McCormack, B.J., Smirnoff, N. and Talbot, N.J. 1997. Glycerol generates
turgor in rice blast. Nature 389: 244-245.
De Lorenzo, G., D'Ovidio, R. and Cervone, F. 2001. The role of polygalacturonaseinhibiting proteins (PGIPs) in defense against pathogenic fungi. Annual Rev.
Phytopathol. 39: 313-335.
Desiderio, A., Aracri, B., Leckie, F., Mattei, B., Salvi, G., Tigelaar, H., Van Roekel,
J.S.C., Baulcombe, D.C., Melchers, L.S. and De Lorenzo, G. 1997.
Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are
expressed in Phaseolus vulgaris. Mol. Plant-Microbe Interact. 10: 852-860.
de Waard, M.A. 1997. Significance of ABC transporters in fungicide sensitivity and
resistance. Pesticide Science 51: 271-275.
Diaz, J., ten Have, A. and van Kan, J.A.L. 2002. The role of ethylene and wound
signaling in resistance of tomato to Botrytis cinerea. Plant Physiol. 129: 1341-1351.
Dickman, M.B. and Mitra, A. 1992. Arabidopsis thaliana as a model for studying
Sclerotinia sclerotiorum pathogenesis. Physiol. Mol. Plant Pathol. 41: 255-263.
Doss, R.P. 1999. Composition and enzymatic activity of the extracellular matrix secreted
by germlings of Botrytis cinerea. Appl. Environm. Microbiol. 65: 404-408.
Doss, R.P., Potter, S.W., Chastagner, G.A. and Christian, J.K. 1993. Adhesion of
nongerminated Botrytis cinerea conidia to several substrata. Appl. Environm.
Microbiol. 59: 1786-1791.
Doss, R.P., Potter, S.W., Soeldner, A.H., Christian, J.K. and Fukunaga, L.E. 1995.
Adhesion of germlings of Botrytis cinerea. Appl. Environm. Microbiol. 61: 260-265.
Dutton, M.V. and Evans, C.S. 1996. Oxalate production by fungi: its role in pathogenicity
and ecology in the soil environment. Can. J. Microbiol. 42: 881-895.
Edlich, W., Lorenz, G., Lyr, H., Nega, E. and Pommer, E.H. 1989. New aspects on the
infection mechanism of Botrytis cinerea Pers. Neth. J. Plant Pathol. 95: 53-62.
Elad, Y. 1992. The use of antioxidants (free radical scavengers) to control grey mould
(Botrytis cinerea) and white mould (Sclerotinia sclerotiorum) in various crops. Plant
Pathol. 41: 417-426.
Elad, Y. and Volpin, H. 1988. The involvement of ethylene, and calcium in gray mold of
pelargonium, Ruscus, and rose plants. Phytoparasitica 16: 119-132.
Ferrari, S., Vairo, D., Ausubel, F.M., Cervone, F. and De Lorenzo, G. 2003. Tandemly
duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are
regulated coordinately by different signal transduction pathways in response to fungal
infection.
Flaishman, M.A. and Kolattukudy, P.E. (1994) Timing of fungal invasion using host's
ripening hormone as a signal. Proc. Natl. Acad. Sci. USA 91: 6579-6583.
Gentile, A.C. 1954. Carbohydrate metabolism and oxalic acid synthesis by Botrytis
cinerea. Plant Physiol. 29: 257-261.
Germeier, C., Hedke, K. and Von Tiedemann, A. 1994. The use of pH-indicators in
diagnostic media for acid-producing plant pathogens. Z. Pflanzenkrankheiten
Pflanzenschutz 101: 498-507.
Gil-Ad, N.L., Bar-Nun, N. and Mayer, A.M. 2001. The possible function of the glcuan
sheath of Botrytis cinerea: effects on the distribution of enzyme activities. FEMS
Microbiol. Letters 199: 109-113.
Godoy, G., Steadman, J.R., Dickman, M.B. and Dam, R. 1990. Use of mutants to
demonstrate the role of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on
Phaseolus vulgaris. Physiol. Mol. Plant Pathol. 37: 179-191.
Gourgues, M., Brunet-Simon, A., Lebrun, M.H. and Levis, C. 2003. The tetraspanin
BcPls1p is required for appressorium-mediated penetration of Botrytis cinerea into
host plant leaves. Molecular Microbiology, in press.
Govrin, E. and Levine, A. 2000. The hypersensitive response facilitates plant infection by
the necrotrophic pathogen Botrytis cinerea. Current Biol. 10: 751-757.
Hain, R., Reif, H.J., Krause, E., Langebartels, R., Kindl, H., Vornam, B., Wiese, W.,
Schmelzer, E. and Schreier, P.H. 1993. Disease resistance results from foreign
85
Muckenschnabel, I., Williamson, B., Goodman, B.A., Lyon, G.D., Stewart, D. and
Deighton, N. 2001b. Markers for oxidative stress associated with soft rots in French
beans (Phaseolus vulgaris) infected by Botrytis cinerea. Planta 212: 376-381.
Muckenschnabel, I., Goodman, B.A., Williamson, B., Lyon, G.D. and Deighton, N. 2002.
Infection of leaves of Arabidopsis thaliana by Botrytis cinerea: Changes in ascorbic
acid, free radicals and lipid peroxidation products. J. Exp. Botany 53: 207-214.
Muckenschnabel, I., Schulze Gronover, C., Deighton, N., Goodman, B.A., Lyon G.D.,
Stewart D. and Williamson, B. 2003. Oxidative effects in uninfected tissue in leaves
of French bean (Phaseolus vulgaris) containing soft rots caused by Botrytis cinerea. J.
Sci. Food Agric. 83: 507-514.
Osbourn, A.E., Melton, R.E., Wubben, J.P., Flegg, L.M., Oliver, R.P. and Daniels, M.J.
1998. Saponin detoxification and fungal pathogenesis. pp. 309-315 in K. Kohmoto
and O.C. Yoder (eds.), Molecular genetics of host-specific toxins in plant diseases,
Kluwer Academic Publishers, Dordrecht.
Pezet, R. 1998. Purification and characterisation of a 32-kda laccase-like stilbene oxidase
produced by Botrytis cinerea Pers. :Fr. FEMS Microbiology Letters 167: 203-208.
Pezet, R., Pont, V. and Hoang Van, K. 1991. Evidence for oxidative detoxification of
pterostilbene and resveratrol by a laccase-like stilbene oxidase produced by Botrytis
cinerea. Physiol. Mol. Plant Pathol. 39: 441-450.
Powell A.L.T, Van Kan J.A.L., Ten Have A., Visser J., Greve L.C., Bennett A.B. and
Labavitch J.M. 2000. Transgenic expression of pear PGIP in tomato limits fungal
colonization. Mol.Plant-Microbe Interact. 13: 942-950.
Prins, T.W., Wagemakers, C.A.M. and Van Kan, J.A.L. 2000a. Cloning and
characterization of a Glutathione S-Transferase homologue from the plant pathogenic
fungus Botrytis cinerea, Mol Plant Pathol. 1: 169-178
Prins, T.W., Tudzynski, P., Von Tiedemann, A., Tudzynski, B., Ten Have, A., Hansen,
M.E., Tenberge, K. and Van Kan, J.A.L. 2000b. Infection strategies of Botrytis
cinerea and related necrotrophic pathogens. pp.33-64 in J. Kronstad (ed.), Fungal
Pathology, Kluwer Academic Publishers.
Prusky, D. 1996. Pathogen quiescence in post-harvest diseases. Annual Rev. Phytopathol.
34:413-434.
Quidde, T., Osbourn, A.E. and Tudzynski, P. 1998. Detoxification of alpha-tomatine by
Botrytis cinerea. Physiol. Mol. Plant Pathol. 52: 151-165.
Rebordinos, L., Cantoral, J.M., Prieto, M.V., Hanson, J.R. and Collado, I.G. 1996. The
phytotoxic activity of some metabolites of Botrytis cinerea. Phytochemistry 42: 383387.
Reignault, P., Mercier, M., Bompeix, G. and Boccara, M. 1994. Pectin methylesterase
from Botrytis cinerea: Physiological, biochemical and immunochemical studies.
Microbiology 140: 3249-3255.
Salinas, J. and Verhoeff, K. 1995. Microscopical studies of the infection of gerbera
flowers by Botrytis cinerea. Eur. J. Plant Pathol. 101: 377-386.
Salinas, J. C. 1992. Function of cutinolytic enzymes in the infection of gerbera flowers by
Botrytis cinerea, Ph.D.-thesis, University of Utrecht, The Netherlands.
Sbaghi, M., Jeandet, P., Bessis, R. and Leroux, P. 1996. Degradation of stilbene-type
phytoalexins in relation to the pathogenicity of Botrytis cinerea to grapevines. Plant
Pathol. 45: 139-144.
Schoonbeek, H., Del Sorbo, G. and de Waard, M.A. 2001. The ABC transporter BcatrB
affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the
fungicide fenpiclonil. Mol. Plant-Microbe Interact. 14: 562-571.
Schoonbeek, H., Raaijmakers, J. and de Waard, M.A. 2002. Fungal ABC transporters and
microbial interactions in natural environments. Mol. Plant-Microbe Interact. 15: 11651172.
Schouten, A., Tenberge, K.B., Vermeer, J., Stewart, J. Wagemakers, C.A.M., Williamson,
B. and van Kan, J.A.L. 2002a. Functional analysis of an extracellular catalase of
Botrytis cinerea. Mol. Plant Pathol. 3: 227-238.
87
Schouten A., Wagemakers, C.A.M., Stefanato, F., Van der Kaaij R.M. and van Kan,
J.A.L. 2002b. Resveratrol acts as a natural profungicide and induces self-intoxication
by a specific laccase. Mol. Microbiol. 43: 883-894.
Schwacke, R. and Hager, A. (1992) Fungal elicitors induce a transient release of active
oxygen species from cultured spruce cells that is dependent on Ca2+ and proteinkinase activity. Planta 187: 136-141.
Staples, R.C. and Mayer, A.M. 1995. Putative virulence factors of Botrytis cinerea acting
as a wound pathogen. FEMS Microbiol. Letters 134: 1-7.
Tenberge, K.B., Beckedorf, M., Hoppe, B, Schouten, A., Solf, M. and von den Driesch,
M. 2002. In situ localization of AOS in host-pathogen interactions. MIcrosc.
Microanal. 8 (suppl. 2): 250-251.
Ten Have, A., Mulder, W., Visser, J. and van Kan, J.A.L. 1998. The endopolygalacturonase gene Bcpg1 is required for full virulence of Botrytis cinerea. Mol.
Plant-Microbe Interact. 11: 1009-1016.
Ten Have, A., Wubben, J.P., Oude-Breuil, W., Mulder, W., Visser, J. and van Kan, J.A.L.
2001. Botrytis cinerea endopolygalacturonase genes are differentially expressed in
various plant tissues. Fungal Genet. Biol. 33: 97-105.
Ten Have, A., Tenberge, K.B., Benen, J.A.E., Tudzynski, P., Visser, J. and van Kan,
J.A.L. 2002. The contribution of cell wall degrading enzymes to pathogenesis of
fungal plant pathogens. pp. 341-358 in F. Kempken (ed.), The Mycota XI,
Agricultural applications, Springer Verlag, Berlin/Heidelberg/New York.
Thomzik, J.E., Stenzel, K., Stocker, R., Schreier, P.H., Hain, R., and Stahl, D.J. (1997)
Synthesis of a grapevine phytoalexin in transgenic tomatoes (Lycopersicon
esculentum mill.) conditions resistance against Phytophthora infestans. Physiol. Mol.
Plant Pathol.51: 265-278.
Valette-Collet, O., Cimerman, A., Reignault, P., Levis, C. and Boccara, M. 2003.
Disruption of Botrytis cinerea pectin methylesterase gene reduces virulence on several
host plants. Mol. Plant-Microbe Interact. 16: 360-367.
van den Heuvel, J. 1981. Effect of inoculum composition on infection of French bean
leaves by conidia of Botrytis cinerea. Neth. J. Plant Pathol. 87: 55-64.
van den Heuvel, J. and Waterreus, L.P. 1983. Conidial concentration as an important
factor determining the type of infection structures formed by Botrytis cinerea on
leaves of French bean (Phaseolus vulgaris). Plant Pathol. 32: 263-272.
van der Vlugt-Bergmans, C.J.B., Wagemakers, C.A.M. and van Kan, J.A.L. 1997a.
Cloning and expression of the cutinase A gene of Botrytis cinerea. Mol. PlantMicrobe Interact. 10: 21-29.
van der Vlugt-Bergmans, C.J.B., Wagemakers, C.A.M., Dees, D.C.T. and van Kan, J.A.L.
1997b. Catalase A from Botrytis cinerea is not expressed during infection on tomato
leaves. Physiol. Mol. Plant Pathol. 50: 1-15.
van Kan, J.A.L., van 't Klooster, J.W., Wagemakers, C.A.M., Dees, D.C.T. and van der
Vlugt-Bergmans, C.J.B. 1997. Cutinase A of Botrytis cinerea is expressed, but not
essential, during penetration of gerbera and tomato. Mol. Plant-Microbe Interact. 10:
30-38.
Verhoeff, K. and Liem, J.I. 1975. Toxicity of tomatine to Botrytis cinerea, in relation to
latency. Phytopathologische Z. 82: 333-338.
Verhoeff, K., Leeman, M., van Peer, R., Posthuma, L., Schot, N. and van Eijk, G.W.
1988. Changes in pH and the production of organic acids during colonisation of
tomato petioles by Botrytis cinerea. J. Phytopathol. 122: 327-336.
Von Tiedemann, A. 1997. Evidence for a primary role of active oxygen species in
induction of host cell death during infection of leaves with Botrytis cinerea. Physiol.
Mol. Plant Pathol. 50: 151-166.
Weigend, M. and Lyr, H. 1996. The involvement of oxidative stress in the pathogenesis
of Botrytis cinerea on Vicia faba leaves. Z. Pflanzenkrankheiten Pflanzenschutz 103:
310-320.
Williamson, B., Duncan, G.H., Harrison, J.G., Harding, L.A., Elad, Y. and Zimand, G.
88
Figure
Spore attachment
Germination
Appressorium
Evasion
Primary lesion formation
of host
defence
responses
(Quiescence)
Lesion expansion,
tissue maceration
Sporulation
Fig. 1. Different stages in the infection process of B. cinerea. The shaded box represents
the host tissue.
89
90